Histidine kinase

ABSTRACT

The invention provides histidine kinase polypeptides and polynucleotides encoding histidine kinase polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing histidine kinase polypeptides to screen for antibacterial compounds.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the histidine kinasefamily, as well as their variants, herein referred to as “histidinekinase,” “histidine kinase polynucleotide(s),” and “histidine kinasepolypeptide(s)” as the case may be.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics The Staphylococcimake up a medically important genera of microbes. They are known toproduce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thromboplebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past few decades. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains that are resistant to some or all of thestandard antibiotics. This phenomenon has created an unmet medical needand demand for new anti-microbial agents, vaccines, drug screeningmethods, and diagnostic tests for this organism.

Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces “functional genomics,” that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on “positional cloning” and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the histidine kinase embodiments of the invention, That have apresent benefit of, among other things, being useful to screen compoundsfor antimicrobial activity. Such factors are also useful to determinetheir role in pathogenesis of infection, dysfunction and disease. Thereis also a need for identification and characterization of such factorsand their antagonists and agonists to find ways to prevent, ameliorateor correct such infection, dysfunction and disease.

SUMMARY OF THE INVENTION

The present invention relates to histidine kinase, in particularhistidine kinase polypeptides and histidine kinase polynucleotides,recombinant materials and methods for their production. In anotheraspect, the invention relates to methods for using such polypeptides andpolynucleotides, including treatment of microbial diseases, amongstothers. In a further aspect, the invention relates to methods foridentifying agonists and antagonists using the materials provided by theinvention, and for treating microbial infections and conditionsassociated with such infections with the identified agonist orantagonist compounds. In a still further aspect, the invention relatesto diagnostic assays for detecting diseases associated with microbialinfections and conditions associated with such infections, such asassays for detecting histidine kinase expression or activity.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to histidine kinase polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a histidinekinase of Staphylococcus aureus, that is related by amino acid sequencehomology to ResE protein from B.subtilis polypeptide. The inventionrelates especially to histidine kinase having a nucleotide and aminoacid sequences set out in Table 1 as SEQ ID NO:1 and SEQ ID NO:2respectively. Note that sequences recited in the Sequence Listing belowas “DNA” represent an exemplification of the invention, since those ofordinary skill will recognize that such sequences can be usefullyemployed in polynucleotides in general, including ribopolynucleotides.

TABLE 1 histidine kinase Polynucleotide and Polypeptide Sequences (A)Staphylococcus aureus histidine kinase polynucleotide sequence [SEQ IDNO:1] 5′-    1 AGAAAAGTTA AATCGTGTGT CTAGCGAAGC TGCGCATATG ATTCAAACAG 51TCTGGGGCGT TGGGTATAAA TTTGAGGTTA AATCTAATGA TGAGCCGGCT 101 AAATAGTGTCGTAATTAAAC TGTGGTTAAC TATTATTTTA ATAGTGACGA 151 CAGTTTTAAT TTTATTAAGTATTGCTTTAA TTACCTTTAT GCAATACTAT 201 TTCACACAAG AAACCGAAAA TGCCATAAGAGAAGATGCTA GACGTATAAG 251 TTCACTGGTC GAACAATCAC ATAATAAAGA AGAAGCAATAAAATATAGTC 301 AAACATTAAT TGAAAATCCT GGTGGGTTGA TGATTATAAA TAATAAACAT351 CGTCAATCAA CGGCTTCACT TTCTAATATT AAAAAGCAAA TGTTGAATGA 401AGTAGTCAAC AACGACCATT TTGACGATGT GTTTGATAAA GGTAAATCTG 451 TTACTCGAAATGTAACGATT AAAGAAAAGG GTTCATCTCA AACATATATT 501 TTGTTAGGCT ATCCAACAAAAGCACAGAAG AATAGTCATA GCAAATATAG 551 TGGAGTCTTT ATATATAAAG ACTTGAAATCAATCGAAGAT ACAAATAATG 601 CTATTACGAT TATCACTATA ATTACGGCTG TTATTTTCTTAACAATTACA 651 ACAGTCTTTG CGTTTTTCTT ATCGTCAAGA ATTACAAAAC CTTTAAGACG701 TTTAAGAGAC CAAGCTACAC GTGTATCTGA AGGGGATTAC TCTTATAAAC 751CTTCTGTCAC AACGAAAGAT GAAATTGGTC AATTATCGCA GGCATTTAAT 801 CAGATGAGTACAGAAATCGA AGAGCATGTC GACGCATTAT CCACATCTAA 851 AAATATTAGA GACAGCTTAATTAACTCTAT GGTAGAAGGT GTCCTAGGTA 901 TTAATGAGAG TCGACAAATT ATCTTATCTAATAAGATGGC GAATGATATT 951 ATGGACAATA TTGATGAAGA TGCTAAAGCT TTCTTATTAAGACAAATTGA 1001 AGATACTTTT AAATCAAAAC AAACGGAAAT GCGTGATTTA GAAATGAATG1051 CACGATTCTT TGTTGTGACC ACAAGCTATA TCGATAAGAT TGAACAGGGA 1101GGTAAAAGTG GTGTTGTTGT GACAGTTCGT GATATGACTA ATGAGCACAA 1151 TCTAGATCAAATGAAGAAAG ATTTCATTGC TAATGTATCA CATGAATTAC 1201 GAACACCTAT ATCATTACTACAAGGTTATA CTGAATCAAT TGTAGATGGT 1251 ATTGTTACAG AACCGGATGA AATAAAAGAATCGCTCGCCG TTGTCCTTGA 1301 TGAATCGAAA CGTTTAAATC GATTAGTTAA TGAATTGTTAAATGTCGCAC 1351 GCATGGATGC TGAAGGGTTA TCCGTAAATA AAGAAGTTCA GCCTATTGCA1401 GCGTTACTAG ATAAGATGAA AATTAAGTAT CGCCAACAAG CTGATGATTT 1451AGGTCTAAAT ATGACTTTTA ATTATTGTAA GAAGCGTGTT TGGAGTTATG 1501 ATATGGATCGCATGGACCAA GTACTAACGA ACTTAATTGA TAATGCATCA 1551 CGTTATACGA AACCTGGAGATGAAATTGCA ATTACTTGTG ATGAAAATGA 1601 AAGCGAAGAT ATTTTATACA TTAAAGATACAGGCACAGGC ATTGCACCAG 1651 AACATTTACA ACAAGTATTT GATCGTTTTT ATAAAGTTGATGCAGCGAGA 1701 ACGCGAGGTA AACAAGGTAC CGGTTTAGGT TTGTTCATTT GTAAAATGAT1751 TATCGAAGAG CATGGTGGTT CCATTGATGT TAAAAGCGAA TTAGGAAAAG 1801GCACAACATT TATTATTAAA CTACCAAAAC CAGAATAAAA CTGAATATAG 1851 TTATTTAAGAACGCATGTTA TTGATTAGAG ACTCTAATTT ATAGCATGCG 1901 TTTTTTGATT GATGTCGAAAGTTTTGTAAG TGGATTAGGA TTAGGGTTTT 1951 TGCGAATATC AACTATTAAA TATATTACTAATTTATATAA AAATATAAAG 2001 TTTGATAAAG TTATTTATTT GATTATAAAA ATAGGGTAAAATATAGATAT 2051 ATTGTAATAA TTAAATTATT CGAGGTGTCA TATGAAAAAA TTCATTGGAT2101 CAGTTTTAGC TACGACATTA ATTTTAGGGG GATGTTCCAT GATGGAAAAT 2151GAATCAAGTA AAGACACGAA TACAGAAACA AAATCAGTAC CAGAAGAAAT 2201 GGAAGCTTCAAAATATGTAG GACAAGGCTT CCAACCACCT GCAG -3′ (B) Staphylococcus aureushistidine kinase polypeptide sequence deduced from a polynucleotidesequence in this table [SEQ ID NO:2]. NH₂-   1 MMSRLNSVVI KLWLTIILIVTTVLILLSIA LITFMQYYFT QETENAIRED 51 ARRISSLVEQ SHNKEEAIKY SQTLIENPGGLMIINNKHRQ STASLSNIKK 101 QMLNEVVNND HFDDVFDKGK SVTRNVTIKE KGSSQTYILLGYPTKAQKNS 151 HSKYSGVFIY KDLKSIEDTN NAITIITIIT AVIFLTITTV FAFFLSSRIT201 KPLRRLRDQA TRVSEGDYSY KPSVTTKDEI GQLSQAFNQM STEIEEHVDA 251LSTSKNIRDS LINSMVEGVL GINESRQIIL SNKAANDIMD NIDEDAKAFL 301 LRQIEDTFKSKQTEMRDLEM NARFFVVTTS YIDKIEQGGK SGVVVTVRDM 351 TNEHNLDQMK KDFIANVSHELRTPISLLQG YTESIVDGIV TEPDEIKESL 401 AVVLDESKRL NRLVNELLNV ARMDAEGLSVNKEVQPIAAL LDKMKIKYRQ 451 QADDLGLNMT FNYCKKRVWS YDMDRMDQVL TNLIDNASRYTKPGDEIAIT 501 CDENESEDIL YIKDTGTGIA PEHLQQVFDR FYKVDAARTR GKQGTGLGLF551 ICKMIIEEHG GSIDVKSELG KGTTFIIKLP KPE-COOH

Deposited Materials

A deposit comprising a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and Maine BacteriaLtd. (herein “NCIMB”), 23 St. Machar Drive, Aberdeen AB2 1RY, Scotlandon Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, and referred toas Staphylococcus aureus WCUH29 on deposit. The Staphylococcus aureusstain deposit is referred to herein as “the deposited strain” or as “theDNA of the deposited strain.”

The deposited strain comprises a full length histidine kinase gene. Thesequence of the polynucleotides comprised in the deposited stain, aswell as the amino acid sequence of any polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent The deposited stain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStaphylococcus aureus WCUH 29 strain, which polypeptide is comprised inthe deposited strain. Further provided by the invention are histidinekinase polynucleotide sequences in the deposited strain such as DNA andRNA, and amino acid sequences encoded thereby. Also provided by theinvention are histidine kinase polypeptide and polynucleotide sequencesisolated from the deposited strain.

Polypeptides

Histidine kinase polypeptide of the invention is substantiallyphylogenetically related to other proteins of the histidine kinasefamily.

In one aspect of the invention there are provided polypeptides ofStaphylococcus aureus referred to herein as “histidine kinase” and“histidine kinase polypeptides” as well as biologically, diagnostically,prophylactically, clinically or therapeutically useful variants thereof,and compositions comprising the same.

Among the particularly preferred embodiments of the invention arevariants of histidine kinase polypeptide encoded by naturally occurringalleles of a histidine kinase gene. The present invention furtherprovides for an isolated polypeptide that: (a) comprises or consists ofan amino acid sequence that has at least 95% identity, most preferablyat least 97-99% or exact identity, to that of SEQ ID NO:2 over theentire length of SEQ ID NO:2; (b) a polypeptide encoded by an isolatedpolynucleotide comprising or consisting of a polynucleotide sequencethat has at least 95% identity, even more preferably at least 97-99% orexact identity to SEQ ID NO:1 over the entire length of SEQ ID NO:1; (c)a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence encoding a polypeptide that hasat least 95% identity, even more preferably at least 97-99% or exactidentity, to the amino acid sequence of SEQ ID NO:2, over the entirelength of SEQ ID NO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular a mature polypeptide) as well as polypeptidesand fragments, particularly those that has a biological activity ofhistidine kinase, and also those that have at least 95% identity to apolypeptide of Table 1 [SEQ ID NO:2] and also include portions of suchpolypeptides with such portion of the polypeptide generally comprisingat least 30 amino acids and more preferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

X−(R₁)_(m)−(R₂)−(R₃)_(n)−Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStaphylococcus aureus, however, it may preferably be obtained from otherorganisms of the same taxonomic genus. A polypeptide of the inventionmay also be obtained, for example, from organisms of the same taxonomicfamily or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. As with histidine kinase polypeptides,fragments may be “free-standing,” or comprised within a largerpolypeptide of which they form a part or region, most preferably as asingle continuous region in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Staphylococcus aureus, are also preferred. Furtherpreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodehistidine kinase polypeptides, particularly polynucleotides that encodea polypeptide herein designated histidine kinase.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding histidine kinase polypeptidescomprising a sequence set out in Table 1 [SEQ ID NO:1] that includes afull length gene, or a variant thereof. The Applicants believe that thisfull length gene is essential to the growth and/or survival of anorganism that possesses it, such as Staphylococcus aureus.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing histidine kinase polypeptidesand polynucleotides, particularly Staphylococcus aureus histidine kinasepolypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a histidine kinasepolypeptide having a deduced amino acid sequence of Table 1 [SEQ IDNO:2] and polynucleotides closely related thereto and variants thereof

In another particularly preferred embodiment of the invention there is ahistidine kinase polypeptide from Staphylococcus aureus comprising orconsisting of an amino acid sequence of Table 1 [SEQ ID NO:2], or avariant thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding histidine kinase polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Staphylococcus aureus WCUH29 cells as stating material, followed by obtaining a full length clone.For example, to obtain a polynucleotide sequence of the invention, suchas a polynucleotide sequence given in Table 1 [SEQ ID NO:1], typically alibrary of clones of chromosomal DNA of Staphylococcus aureus WCUH 29 inE. coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent hybridization conditions. By sequencingthe individual clones thus identified by hybridization with sequencingprimers designed from the original polypeptide or polynucleotidesequence it is then possible to extend the polynucleotide sequence inboth directions to determine a full length gene sequence. Conveniently,such sequencing is performed, for example, using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Direct genomic DNA sequencing may also be performed toobtain a full length gene sequence. Illustrative of the invention, eachpolynucleotide set out in Table 1 [SEQ ID NO:1] was discovered in a DNAlibrary derived from Staphylococcus aureus WCUH 29.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth im Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 87 and the stopcodon that begins at nucleotide number 1836 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

In a further a the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynucleotidesequence that has at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ IDNO:1, or the entire length of that portion of SEQ ID NO:1 which encodesSEQ ID NO:2; (b) a polynucleotide sequence encoding a polypeptide thathas at least 95% identity, even more preferably at least 97-99% or 100%exact, to the amino acid sequence of SEQ ID NO:2, over the entire lengthof SEQ ID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including horthologs and orthologs from species other thanStaphylococcus aureus, may be obtained by a process that comprises thesteps of screening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof; andisolating a full-length gene and/or genomic clones comprising saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least one noncoding sequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize MRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 87 to the nucleotide immediatelyupstream of or including nucleotide 1835 set forth in SEQ ID NO:1 ofTable 1, both of that encode a histidine kinase polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:

X−(R₁)_(m)−(R₂)−(R₃)_(n)−Y

wherein, at the 5′ end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3′ end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5′ end nucleic acid residue is at the left,bound to R₁, and its 3′ end nucleic acid residue is at the right, boundto R₃. Any stretch of nucleic acid residues denoted by either R₁ and/orR₂, where m and/or n is greater than 1, may be either a heteropolymer ora homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, that can be adouble-stranded polynucleotide wherein the formula shows a first strandto which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1and 50, 100 or500,and n is an integer between1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Staphylococcus aureus, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polynucleotide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus histidinekinase having an amino acid sequence set out in Table 1 [SEQ ID NO:2].The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, polynucleotides interrupted by integrated phage, an integratedinsertion sequence, an integrated vector sequence, an integratedtransposon sequence, or due to RNA editing or genomic DNAreorganization) together with additional regions, that also may comprisecoding and/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodinghistidine kinase variants, that have the amino acid sequence ofhistidine kinase polypeptide of Table 1 [SEQ ID NO:2] in which several,a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues aresubstituted, modified, deleted and/or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, that do not alter the properties and activities of histidinekinase polypeptide.

Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nucleicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5′ and/or 3′ end of the polynucleotide sequence of SEQ ID NO:1.

Further preferred embodiments of the invention are polynucleotides thatare at least 95% or 97% identical over their entire length to apolynucleotide encoding histidine kinase polypeptide having an aminoacid sequence set out in Table 1 [SEQ ID NO:2], and polynucleotides thatare complementary to such polynucleotides. Most highly preferred arepolynucleotides that comprise a region that is at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to histidine kinase polynucleotide sequences, suchas those polynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionspecially relates to polynucleotides tat hybridize under stringentconditions to the polynucleotides described herein As herein used, theterms “stringent conditions” and “stringent hybridization conditions”mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (PH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycomprising a complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said polynucleotide sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding histidine kinase and toisolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to a histidine kinasegene. Such probes generally will comprise at least 15 nucleotideresidues or base pairs. Preferably, such probes will have at least 30nucleotide residues or base pairs and may have at least 50 nucleotideresidues or base pairs. Particularly preferred probes will have at least20 nucleotide residues or base pairs and will have lee than 30nucleotide residues or base pairs.

A coding region of a histidine kinase gene may be isolated by screeningusing a DNA sequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from MRNA extracted from a chosen tissue and an‘adaptor’ sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the “missing” 5′ end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is a mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to a mature polypeptide (when amature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from a mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

As will be recognized, the entire polypeptide encoded by an open readingframe is often not required for actity. Accordingly, it has becomeroutine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol available at www.promega.com). The digested endpoints can berepaired (e.g., by ligation to synthetic linkers) to the extentnecessary to preserve an open reading frame. In this way, the nucleicacid of SEQ ID NO:1 readily provides contiguous fragments of SEQ ID NO:2sufficient to provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO:2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells tat are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA construct of the invention.

Recombine polypeptides of the present invention may be prepared byprocesses well known m those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring arbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci staphylococci, enterococci E. coli,streptomyces, cyanobacteria, Bacillus subtilis, and Staphylococcusaureus ; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL,(supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of histidine kinasepolynucleotides and polypeptides of the invention for use as diagnosticreagents. Detection of histidine kinase polynucleotides and/orpolypeptides in a eukaryote, particularly a mammal, and especially ahuman, will provide a diagnostic method for diagnosis of disease,staging of disease or response of an infectious organism to drugs.Eukaryotes, particularly mammals, and especially humans, particularlythose infected or suspected to be infected with an organism comprisingthe histidine kinase gene or protein may be detected at the nucleic acidor amino acid level by a variety of well known techniques as well as bymethods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled histidine kinase polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexesby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be d by alterations in theelectrophoretic mobility of polynucleotide fragments in gels as comparedto a reference sequence. This may be carried out with or withoutdenaturing agents. Polynucleotide differences may also be detected bydirect DNA or RNA sequencing. See, for example, Myers et al., Science,230: 1242 (1985). Sequence changes at specific locations also may berevealed by nuclease protection assays, such as RNase, V1 and S1protection assay or a chemical cleavage method. See, for example, Cottonet al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisinghistidine kinase nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit that comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof, or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2. Itwill be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotype, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding histidine kinase polypeptide can be used toidentify and analyze mutations. The invention further provides theseprimers with 1, 2, 3 or 4 nucleotides removed from the 5′ and/or the 3′end. These primers nay be used for, among other things, amplifyinghistidine kinase DNA and/or RNA isolated from a sample derived from anindividual, such as a body material. The primers may be used to amplifya polynucleotide isolated from an infected individual, such that thepolynucleotide may then be subject to various techniques for elucidationof the polynucleotide sequence. In this way, mutations in thepolynucleotide sequence may be detected and used to diagnose and/orprognose the infection or its stage or course, or to serotype and/orclassify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStaphylococcus aureus, comprising determining from a sample derived froman individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a histidine kinase polynucleotidecan be measured using any on of the methods well known in the art forthe quantitation of polynucleotides, such as, for example,amplification, PCR, RT-PCR, RNase protection, Northern blotting,spectrometry and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of histidine kinase polypeptide compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a histidine kinase polypeptide, in a sample derived from ahost, such as a bodily material, are well-known to those of skill im theart. Such assay methods include radioimmunoassays, competitive-bidingassays, Western Blot analysis, antibody sandwich assays, antibodydetection and ELISA assays.

Antagonists and Agonists—Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the biding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g.,inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g., inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, ofhistidine kinase polypeptides and polynucleotides; or may be structuralor functional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

The screening methods may simply measure the biding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists, in the absence of an agonist or antagonist, by testing whetherthe candidate compound results in inhibition of activation of thepolypeptide or polynucleotide, as the case may be. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution comprising a polypeptide or polynucleotide ofthe present invention, to form a mixture, measuring histidine kinasepolypeptide and/or polynucleotide activity in the mixture, and comparingthe histidine kinase polypeptide and/or polynucleotide activity of themixture to a standard. Fusion proteins, such as those made from Fcportion and histidine kinase polypeptide, as herein described, can alsobe used for high-throughput screening assays to identify antagonists ofthe polypeptide of the present invention, as well as of phylogeneticallyand and/or functionally related polypeptides (see D. Bennett et al., JMol Recognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of MRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentsthat may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose that enhance (agonist) or block (antagonist) the action ofhistidine kinase polypeptides or polynucleotides, particularly thosecompounds that are bacteristatic and/or bactericidal. The method ofscreen may involve high-throughput techniques. For example, to screenfor agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising histidine kinase polypeptide anda labeled substrate or ligand of such polypeptide is incubated in theabsence or the presence of a candidate molecule that may be a histidinekinase agonist or antagonist. The ability of the candidate molecule toagonize or antagonize the histidine kinase polypeptide is reflected indecreased binding of the labeled ligand or decreased production ofproduct from such substrate. Molecules that bind gratuitously, i.e.,without inducing the effects of histidine kinase polypeptide are mostlikely to be good antagonists. Molecules that bind well and, as the casemay be, increase the rate of product production from substrate, increasesignal transduction, or increase chemical channel activity are agonists.Detection of the rate or level of, as the case may be, production ofproduct from substrate, signal transduction, or chemical channelactivity may be enhanced by using a reporter system. Reporter systemsthat may be useful in this regard include but are not limed tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in histidine kinase polynucleotide orpolypeptide activity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance,¹²⁵¹I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identityagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by histidine kinase polypeptide associatingwith another histidine kinase polypeptide or other polypeptide, labeledto comprise a fluorescently-labeled molecule will have higherpolarization values than a fluorescently labeled monomeric protein. Itis preferred that this method be used to characterize small moleculesthat disrupt polypeptide complexes.

Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of histidine kinasepolypeptide dimers, trimers, tetramers or higher order structures, orstructures formed by histidine kinase polypeptide bound to anotherpolypeptide. histidine kinase polypeptide can be labeled with both adonor and acceptor fluorophore. Upon mixing of the two labeled speciesand excitation of the donor fluorophore, fluorescence energy transfercan be detected by observing fluorescence of the acceptor. Compoundsthat block dimerization will inhibit fluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on histidine kinase polypeptide self-association as well as anassociation of histidine kinase polypeptide and another polypeptide orsmall molecule. Histidine kinase polypeptide can be coupled to a sensorchip at low site density such that covalently bound molecules will bemonomeric. Solution protein can then passed over the histidine kinasepolypeptide -coated surface and specific binding can be detected inreal-time by monitoring the change in resonance angle caused by a changeim local refractive index. This technique can be used to characterizethe effect of small molecules on kinetic rates and equilibrium bindingconstants for histidine kinase polypeptide self-association as well asan association of histidine kinase polypeptide and another polypeptideor small molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of histidine kinase polypeptide withanother histidine kinase polypeptide or a different polypeptide.Histidine kinase polypeptide can be coupled to a scintillation-filledbead. Addition of radio-labeled histidine kinase polypeptide results inbinding where the radioactive source molecule is in close proximity tothe scintillation fluid. Thus, signal is emitted upon histidine kinasepolypeptide binding and compounds that prevent histidine kinasepolypeptide self-association or an association of histidine kinasepolypeptide and another polypeptide or small molecule will diminishsignal.

In other embodiments of the invention there are provided methods foridentifying compounds that bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contact a polypeptide and/orpolynucleotide of the invention with a compound to be screened underconditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for histidine kinase agonists is acompetitive assay that combines histidine kinase and a potential agonistwith histidine kinase-binding molecules, recombinant histidine kinasebinding molecules, natural substrate or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. Histidine kinase can be labeled, such as by radioactivity or acolorimetric compound, such that the number of histidine kinasemolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist or antagonist of thepolypeptide and/or polynucleotide, by: (a) determining in the firstinstance the three-dimensional structure of the polypeptide and/orpolynucleotide, or complexes thereof; (b) deducing the three-dimensionalstructure for the likely reactive site(s), binding site(s) or motif(s)of an agonist or antagonist; (c) synthesizing candidate compounds thatare predicted to bind to or react with the deduced binding site(s),reactive site(s), and/or motif(s); and (d) testing whether the candidatecompounds are indeed agonists or antagonists.

It will be further appreciated that this will normally be an iterativeprocess, and this iterative process may be performed using automated andcomputer-controlled steps.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of histidine kinase polypeptide and/orpolynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises admit to an individual in need thereof an inhibitorcompound (antagonist) as herein described, optionally in combinationwith a pharmaceutically acceptable carrier, in an amount effective toinhibit the function and/or expression of the polypeptide and/orpolynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the histidinekinase polypeptide and/or polypeptide.

In still another approach, expression of the gene encoding endogenoushistidine kinase polypeptide can be inhibited using expression blockingtechniques. This blocking may be targeted against any step in geneexpression, but is preferably targeted against transcription and/ortranslation. An examples of a known technique of this sort involve theuse of antisense sequences, either internally generated or separatelyadministered (see, for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides thatform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial histidine kinase proteins that mediate tissue damage and/or;to block the normal progression of pathogenesis in infections initiatedother than by the implantation of in-dwelling devices or by othersurgical techniques.

In accordance with yet another aspect of the invention, there areprovided histidine kinase agonists and antagonists, preferablybacteristatic or bactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein “H. pylori”) bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofhistidine kinase polypeptides and/or polynucleotides) found usingscreens provided by the invention, or known in the art, particularlynarrow-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also prevent, inhibit and/or cure gastric ulcers andgastritis.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

“Disease(s)” means any disease caused by or related to infection by abacteria, including, for example, disease, such as, infections of theupper respiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, tyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic abscess, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthritis,osteomyelitis).

“Host cell(s)” is a cell that has been introduced (e.g., transformed ortransfected) or is capable of introduction (e.g., transformation ortransfection) by an exogenous polynucleotide sequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J Molec. Biol 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty:12

Gap Length Penalty:4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty:50

Gap Length Penalty:3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for “identity” for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:

n _(n) ≦x _(n)−(x _(n) ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 95, 97 or 100% identity to apolypeptide reference sequence of SEQ ID NO:2, wherein said polypeptidesequence may be identical to the reference sequence of SEQ ID NO:2 ormay include up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of amino acids in SEQ ID NO:2, or:

n _(a) ≦x _(a)−(x _(a) ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

“Individual(s)” means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

“Organism(s)” means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a member of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double- strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA tat serve many usefulpurposes known to those of skill in the art. The term“polynucleotide(s)” as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells.“Polynucleotide(s)” also embraces short polynucleotides often referredto as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commnonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Cry, W. H. Freeman and Company,New York (1993) and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in POSTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York(1983); Seifter et al., Meth Enzymol 182:626-646 (1990) and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62 (1992). Polypeptides may be branched orcyclic, with or without branching. Cyclic, branched and branchedcircular polypeptides may result from post-translational naturalprocesses and may be made by entirely synthetic methods, as well.

“Recombinant expression system(s)” refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

“Variant(s)” as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1- 3,1- 2or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, that arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones comprising overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 Below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E.coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., Rsal, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

2 1 2244 DNA Staphylococcus aureus 1 agaaaagtta aatcgtgtgt ctagcgaagctgcgcatatg attcaaacag tctggggcgt 60 tgggtataaa tttgaggtta aatctaatgatgagccggct aaatagtgtc gtaattaaac 120 tgtggttaac tattatttta atagtgacgacagttttaat tttattaagt attgctttaa 180 ttacctttat gcaatactat ttcacacaagaaaccgaaaa tgccataaga gaagatgcta 240 gacgtataag ttcactggtc gaacaatcacataataaaga agaagcaata aaatatagtc 300 aaacattaat tgaaaatcct ggtgggttgatgattataaa taataaacat cgtcaatcaa 360 cggcttcact ttctaatatt aaaaagcaaatgttgaatga agtagtcaac aacgaccatt 420 ttgacgatgt gtttgataaa ggtaaatctgttactcgaaa tgtaacgatt aaagaaaagg 480 gttcatctca aacatatatt ttgttaggctatccaacaaa agcacagaag aatagtcata 540 gcaaatatag tggagtcttt atatataaagacttgaaatc aatcgaagat acaaataatg 600 ctattacgat tatcactata attacggctgttattttctt aacaattaca acagtctttg 660 cgtttttctt atcgtcaaga attacaaaacctttaagacg tttaagagac caagctacac 720 gtgtatctga aggggattac tcttataaaccttctgtcac aacgaaagat gaaattggtc 780 aattatcgca ggcatttaat cagatgagtacagaaatcga agagcatgtc gacgcattat 840 ccacatctaa aaatattaga gacagcttaattaactctat ggtagaaggt gtcctaggta 900 ttaatgagag tcgacaaatt atcttatctaataagatggc gaatgatatt atggacaata 960 ttgatgaaga tgctaaagct ttcttattaagacaaattga agatactttt aaatcaaaac 1020 aaacggaaat gcgtgattta gaaatgaatgcacgattctt tgttgtgacc acaagctata 1080 tcgataagat tgaacaggga ggtaaaagtggtgttgttgt gacagttcgt gatatgacta 1140 atgagcacaa tctagatcaa atgaagaaagatttcattgc taatgtatca catgaattac 1200 gaacacctat atcattacta caaggttatactgaatcaat tgtagatggt attgttacag 1260 aaccggatga aataaaagaa tcgctcgccgttgtccttga tgaatcgaaa cgtttaaatc 1320 gattagttaa tgaattgtta aatgtcgcacgcatggatgc tgaagggtta tccgtaaata 1380 aagaagttca gcctattgca gcgttactagataagatgaa aattaagtat cgccaacaag 1440 ctgatgattt aggtctaaat atgacttttaattattgtaa gaagcgtgtt tggagttatg 1500 atatggatcg catggaccaa gtactaacgaacttaattga taatgcatca cgttatacga 1560 aacctggaga tgaaattgca attacttgtgatgaaaatga aagcgaagat attttataca 1620 ttaaagatac aggcacaggc attgcaccagaacatttaca acaagtattt gatcgttttt 1680 ataaagttga tgcagcgaga acgcgaggtaaacaaggtac cggtttaggt ttgttcattt 1740 gtaaaatgat tatcgaagag catggtggttccattgatgt taaaagcgaa ttaggaaaag 1800 gcacaacatt tattattaaa ctaccaaaaccagaataaaa ctgaatatag ttatttaaga 1860 acgcatgtta ttgattagag actctaatttatagcatgcg ttttttgatt gatgtcgaaa 1920 gttttgtaag tggattagga ttagggtttttgcgaatatc aactattaaa tatattacta 1980 atttatataa aaatataaag tttgataaagttatttattt gattataaaa atagggtaaa 2040 atatagatat attgtaataa ttaaattattcgaggtgtca tatgaaaaaa ttcattggat 2100 cagttttagc tacgacatta attttagggggatgttccat gatggaaaat gaatcaagta 2160 aagacacgaa tacagaaaca aaatcagtaccagaagaaat ggaagcttca aaatatgtag 2220 gacaaggctt ccaaccacct gcag 2244 2583 PRT Staphylococcus aureus 2 Met Met Ser Arg Leu Asn Ser Val Val IleLys Leu Trp Leu Thr Ile 1 5 10 15 Ile Leu Ile Val Thr Thr Val Leu IleLeu Leu Ser Ile Ala Leu Ile 20 25 30 Thr Phe Met Gln Tyr Tyr Phe Thr GlnGlu Thr Glu Asn Ala Ile Arg 35 40 45 Glu Asp Ala Arg Arg Ile Ser Ser LeuVal Glu Gln Ser His Asn Lys 50 55 60 Glu Glu Ala Ile Lys Tyr Ser Gln ThrLeu Ile Glu Asn Pro Gly Gly 65 70 75 80 Leu Met Ile Ile Asn Asn Lys HisArg Gln Ser Thr Ala Ser Leu Ser 85 90 95 Asn Ile Lys Lys Gln Met Leu AsnGlu Val Val Asn Asn Asp His Phe 100 105 110 Asp Asp Val Phe Asp Lys GlyLys Ser Val Thr Arg Asn Val Thr Ile 115 120 125 Lys Glu Lys Gly Ser SerGln Thr Tyr Ile Leu Leu Gly Tyr Pro Thr 130 135 140 Lys Ala Gln Lys AsnSer His Ser Lys Tyr Ser Gly Val Phe Ile Tyr 145 150 155 160 Lys Asp LeuLys Ser Ile Glu Asp Thr Asn Asn Ala Ile Thr Ile Ile 165 170 175 Thr IleIle Thr Ala Val Ile Phe Leu Thr Ile Thr Thr Val Phe Ala 180 185 190 PhePhe Leu Ser Ser Arg Ile Thr Lys Pro Leu Arg Arg Leu Arg Asp 195 200 205Gln Ala Thr Arg Val Ser Glu Gly Asp Tyr Ser Tyr Lys Pro Ser Val 210 215220 Thr Thr Lys Asp Glu Ile Gly Gln Leu Ser Gln Ala Phe Asn Gln Met 225230 235 240 Ser Thr Glu Ile Glu Glu His Val Asp Ala Leu Ser Thr Ser LysAsn 245 250 255 Ile Arg Asp Ser Leu Ile Asn Ser Met Val Glu Gly Val LeuGly Ile 260 265 270 Asn Glu Ser Arg Gln Ile Ile Leu Ser Asn Lys Met AlaAsn Asp Ile 275 280 285 Met Asp Asn Ile Asp Glu Asp Ala Lys Ala Phe LeuLeu Arg Gln Ile 290 295 300 Glu Asp Thr Phe Lys Ser Lys Gln Thr Glu MetArg Asp Leu Glu Met 305 310 315 320 Asn Ala Arg Phe Phe Val Val Thr ThrSer Tyr Ile Asp Lys Ile Glu 325 330 335 Gln Gly Gly Lys Ser Gly Val ValVal Thr Val Arg Asp Met Thr Asn 340 345 350 Glu His Asn Leu Asp Gln MetLys Lys Asp Phe Ile Ala Asn Val Ser 355 360 365 His Glu Leu Arg Thr ProIle Ser Leu Leu Gln Gly Tyr Thr Glu Ser 370 375 380 Ile Val Asp Gly IleVal Thr Glu Pro Asp Glu Ile Lys Glu Ser Leu 385 390 395 400 Ala Val ValLeu Asp Glu Ser Lys Arg Leu Asn Arg Leu Val Asn Glu 405 410 415 Leu LeuAsn Val Ala Arg Met Asp Ala Glu Gly Leu Ser Val Asn Lys 420 425 430 GluVal Gln Pro Ile Ala Ala Leu Leu Asp Lys Met Lys Ile Lys Tyr 435 440 445Arg Gln Gln Ala Asp Asp Leu Gly Leu Asn Met Thr Phe Asn Tyr Cys 450 455460 Lys Lys Arg Val Trp Ser Tyr Asp Met Asp Arg Met Asp Gln Val Leu 465470 475 480 Thr Asn Leu Ile Asp Asn Ala Ser Arg Tyr Thr Lys Pro Gly AspGlu 485 490 495 Ile Ala Ile Thr Cys Asp Glu Asn Glu Ser Glu Asp Ile LeuTyr Ile 500 505 510 Lys Asp Thr Gly Thr Gly Ile Ala Pro Glu His Leu GlnGln Val Phe 515 520 525 Asp Arg Phe Tyr Lys Val Asp Ala Ala Arg Thr ArgGly Lys Gln Gly 530 535 540 Thr Gly Leu Gly Leu Phe Ile Cys Lys Met IleIle Glu Glu His Gly 545 550 555 560 Gly Ser Ile Asp Val Lys Ser Glu LeuGly Lys Gly Thr Thr Phe Ile 565 570 575 Ile Lys Leu Pro Lys Pro Glu 580

What is claimed is:
 1. An isolated polynucleotide segment comprising afirst polynucleotide sequence or the full complement of the entirelength of the first polynucleotide sequence, wherein the firstpolynucleotide sequence is identical to a reference sequence comprisingnucleotides 87 to 1835 set forth in SEQ ID NO:1, except that, over theentire length corresponding to the reference sequence, n_(n) nucleotidesare substituted, inserted or deleted, wherein n_(n) satisfies thefollowing expression n _(n) ≦x _(n)−(x _(n) ·y) wherein x_(n) is thetotal number of nucleotides in the reference sequence, y is 0.95, andwherein any non-integer product of x_(n) and y is rounded down to thenearest integer before subtracting the product from x_(n); and whereinthe first polynucleotide sequence detects Staphylococcus aureus.
 2. Avector comprising the isolated polynucleotide segment of claim
 1. 3. Anisolated host cell comprising the vector of claim
 2. 4. The isolatedpolynucleotide segment of claim 1, wherein y is 0.97.
 5. The isolatedpolynucleotide segment of claim 1, wherein y is 0.99.
 6. The isolatedpolynucleotide segment of claim 1, wherein the first polynucleotidesequence comprises nucleotides 87 to 1835 set forth in SEQ ID NO:1.
 7. Avector comprising the isolated polynucleotide segment of claim
 6. 8. Anisolated host cell comprising the vector of claim
 7. 9. A polynucleotidewhich encodes a fusion polypeptide and which includes the isolatedpolynucleotide segment of claim
 6. 10. An isolated polynucleotidesegment, comprising a first polynucleotide sequence or the fillcomplement of the entire length of the first polynucleotide sequence,wherein the first polynucleotide sequence hybridizes to the fullcomplement of a reference sequence comprising nucleotides 87 to 1835 setforth in SEQ ID NO:1, wherein the hybridization conditions includeincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 micrograms/mldenatured, sheared salmon sperm DNA, followed by washing in 0.1×SSC atabout 65° C.; and, wherein the first polynucleotide sequence isidentical to the reference sequence, except that, over the entire lengthcorresponding to the reference sequence, n_(n) nucleotides aresubstituted, inserted or deleted, wherein n_(n) satisfies the followingexpression n _(n) ≦x _(n)−(x _(n) ·y) wherein x_(n) is the total numberof nucleotides in the reference sequence, y is 0.95, and wherein anynon-integer product of x_(n) and y is rounded down to the nearestinteger before subtracting the product from x_(n); and wherein the firstpolynucleotide sequence detects Staphylococcus aureus.
 11. The isolatedpolynucleotide segment of claim 10, wherein y is 0.97.
 12. An isolatedpolynucleotide segment comprising a first polynucleotide sequence or thefull complement of the entire length of the first polynucleotidesequence, wherein the first polynucleotide sequence encodes apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2.13. A vector comprising the isolated polynucleotide segment of claim 12.14. An isolated host cell comprising the vector of claim
 13. 15. Apolynucleotide which encodes a fusion polypeptide and which includes theisolated polynucleotide segment of claim
 12. 16. An isolatedpolynucleotide segment comprising a first polynucleotide sequence or thefall complement of the entire length of the first polynucleotidesequence, wherein the first polynucleotide sequence encodes apolypeptide consisting of the amino acid sequence set forth in SEQ IDNO:2.
 17. A vector comprising the isolated polynucleotide segment ofclaim
 16. 18. An isolated host cell comprising the vector of claim 17.